PMC

(A) Left - Etoposide-treated HOSE cells were monitored for apoptosis by caspase-3 activation. Middle - Quantitative RT-PCR shows a moderate increase in SALL2 and a large decrease in c-MYC expression following etoposide treatment. Right - ChIP followed by qRT-PCR shows increased binding of p150 to the c-MYC promoter in etoposide-treated cells. All histograms are based on triplicate determinations. ** denotes p<0.01 comparing means of experimental and control.

(A) OVCA - ovarian serous cystadenocarcinoma. (B) GBM – glioblastoma multiforme. (C ) BRCA – breast invasive carcinoma. (D) LUSC – lung squamous cell carcinoma. n = number of samples; r = Pearson correlation coefficient; p = p-value of the correlation. The lines shown are those of best fit.

(A) Left - overexpression of p150 results in reduced c-MYC expression in HOSE cells shown by quantitative RT PCR. Right – immunoblotting for c-MYC. (B) Left - siRNA-knockdown of endogenous SALL2 in HOSE cells leads to increased c-MYC expression by quantitative RT PCR. Right - immunoblotting for c-MYC. (C) Left - Human c-MYC promoter region and luciferase reporters used in promoter activity assays. The NHE region (−142 to −100) including p150 consensus binding sites (underlined) was deleted in the reporter myc-Luc-ΔNHE. Right - siRNA to SALL2 in HOSE cells results in increased expression of myc-Luc but has no effect on myc-Luc-ΔNHE. (D) Expression of exogenous p150 in p150-deficient RMUGS ovarian cancer cells leads to decreased expression of the reporter myc-Luc. All histograms are based on triplicate determinations. * and ** denote p<0.05 and p<0.01, respectively, comparing means of experimental and control.

(A) The GC-rich nuclease hypersensitive element NHE III₁ of the human c-MYC promoter. Consensus binding sites for p150 are underlined. Approximate positions of transcriptional start sites are shown (P1, P2). (B) An electrophoretic mobility shift assay shows binding of GST-p150 to the labeled NHE III₁ and to the consensus binding sequence (CS) GGGTGGG- as control (<$>\raster(75%)="rg1"<$>). 50× molar excess of unlabeled NHE III₁ and CS compete for binding while the unrelated Oct1 olionucleotide does not. (C) Chromatin immunoprecipitation assays with antibodies to the N- and C-terminal portions of p150 show binding of endogenous p150 to the NHE III₁ region of the c-MYC promoter in HOSE cells. (D) SALL2-knockdown leads to reduced binding of p150 to the c-MYC promoter. HOSE cells were treated with SALL2 siRNA or control oligonucleotide and followed by chromatin immunoprecipitation and quantitative RT-PCR assays. The threshold cycle values of immunoprecipitates were normalized to those of 1% input control samples. The human aldolase A promoter region was included as a negative control. Estimates of binding were based on triplicate samples. ** denotes p<0.01 based on Student t test run on HOSE cells treated with SALL2-siRNA or control RNA.